Mechanisms that enforce T tolerance to self-antigens (self-Ags) in the secondary lymphoid organs (periphery) are critical for preventing development of autoimmune diseases, but can also limit the induction of desirable responses to self-Ags, as in the case of tumor vaccines, or to foreign Ags, as in the case of vaccines for pathogens. Much is known about the mechanisms that mediate peripheral T cell tolerance, but many fundamental questions remain, and developing an increased understanding of these processes will lead to an enhanced ability to manipulate tolerance for prevention or treatment of autoimmune diseases. This Program focuses on addressing these questions by examining CD4 and CD8 T cell tolerance mechanisms at the cellular and molecular levels. The Program includes four projects that all focus on tolerance to self-antigens in autoimmune disease models: Project 1. Analysis of peripheral tolerance in vivo (M. Jenkins) Project 2. Functional characterization of anergic T cells (D. Mueller) Project 3. Self reactivity in polyclonal T cell populations (K. Hogquist) Project 4. Mechanisms of self-Ag-induced non-responsiveness in CD8 T cells (M. Mescher) The overall Program goals are to gain new insights into fundamental mechanisms that lead to T cell tolerance and determine how and where these mechanisms act to enforce tolerance to self-antigens in mice and humans. Each project will apply innovative technologies to address these questions. Of particular note, new technology developed under this Program will allow analysis of self-reactive human T cells at a level not previously possible, and is likely to set the stage fr novel clinical trials. We anticipate that novel insights into mechanistic aspects of tolerance induction gained through the proposed studies will lead to new strategies for promoting tolerance in autoimmune diseases or avoiding tolerance during protective or therapeutic vaccination. RELEVANCE: Each project uses autoimmune disease models to study the cellular and molecular mechanisms for peripheral tolerance induction to self-antigens in T cells. Numerous interactions between the projects will provide synergy in achieving the overall Program goals. An Administrative Core will provide budgetary and scientific oversight and Core B (Autoimmune Mouse Core) will support all four of the projects. Project 1: Analysis of peripheral tolerance in vivo Project Leader: Marc Jenkins DESCRIPTION (provided by applicant): Most people do not suffer from autoimmunity despite the production of CD4+ T cells expressing T cell receptors (TCR) specific for self peptide (p):major histocompatibility complex II (MHCII) ligands. Many studies in TCR transgenic mouse models have shown that this is the case because these CD4+ T cells are deleted in the thymus or differentiate into anergic or suppressive regulatory T (Treg) cells in secondary lymphoid organs. Nevertheless, consensus on the relative contributions of these mechanisms to tolerance to all self antigens has not been reached. Fundamental questions therefore remain to be answered such as how efficient is thymic deletion, do anergic T cells exist, is the Treg cell repertoire really enriched for self p:MHCIl-specific cells, and which of these mechanisms fails during autoimmunity? We will answer these questions by studying polyclonal endogenous CD4+ T cells specific for self p:MHCII ligands using a sensitive p:MHCII tetramer-based cell enrichment method. In mice, we will determine whether T cells expressing TCRs with the highest affinities for ubiquitous self p:MHCII ligands are deleted, and whether some T cells specific for p:MHCII ligands derived from peripheral tissue-specific proteins expressed in the thymus under the control of the Autoimmune Regulator (AIRE) escape deletion but become anergic or differentiate into Treg cells in the secondary lymphoid organs. We will attempt to confirm these hypotheses in humans by direct ex vivo tracking of the number, function, and phenotype of insulin or glutamic acid decarboxylase p:MHCII-specific CD4+ T cells from normoglycemic or type 1 diabetic people. If successful, we will have learned how efficient thymic clonal deletion is, whether anergy exists as a tolerance mechanism, and if self-reactive T cell populations are enriched for Treg cells, all within normal polyclonal repertoires. These experiments could set the stage for future clinical trials to determine if self p:MHCII tetramer-based cell enrichment can be used as a tool for early diagnosis of diabetes or to monitor the efficacy of immunotherapy. RELEVANCE: This project focuses on the mechanisms of immune tolerance that prevent CD4+ T cells from causing autoimmunity. It will employ innovative T cell tracking technology to bridge the gap between mechanistic studies in mouse models and application to the human immune system. The approach described in this application could lead to new methods for diagnosing diabetes and monitoring immunotherapy.